For many applications, e.g. in the case of multi-photon excitation, it is necessary to use as a light source a laser that delivers light in the form of short pulses. Because of their design, some of these lasers, e.g. diode lasers, are synchronizable with an external timing source; others such as, for example, fiber lasers or TiSa lasers, are not synchronizable.
It is problematic that the scanning device and the pulsed light source are operated independently of one another.
The invention further relates to a scanning method using a scanning microscope.
German Unexamined Application DE 101 26 286 A1 discloses a method and an apparatus for point-by-point scanning of a sample. The method is characterized by the steps of generating a setpoint signal for each scanned point, and transferring the setpoint signal to a scanning device. In further steps an actual signal is ascertained, from the position of the scanning device, for each scanned point. Also performed are detection of at least one detected signal for each scanned point, calculation of a display signal and an image point position from the actual signal and/or the setpoint signal and the detected signal, and allocation of the display signal to the image point position.
German Unexamined Application DE 197 02 752 A1 refers to a triggering system for a scanner, in particular for a laser scanning microscope, having an oscillating motor for driving an oscillating mirror that serves for linearly oscillating deflection of a ray bundle. A triggering unit is provided for supplying the oscillating motor with an excitation current that is modifiable in terms of triggering frequency, frequency curve, and amplitude. Triggering is accomplished with a function generator that is connected to the triggering unit, and with a measured value transducer for obtaining a sequence of data regarding the deflected positions of the oscillating mirror. The measured value transducer is linked to the function generator via a logic unit for ascertaining correction values for the excitation current. It is thereby possible, with an evaluation of the information made available from the measured value transducer regarding the actual deflected position of the oscillating mirror, to ascertain correction values with the aid of the logic unit. Said values can in turn be used to influence the triggering frequencies outputted by the function generator in such a way that deviations are minimized or entirely eliminated.
European Patent Application EP 0 845 693 A1 discloses a confocal microscope and a method for generating three-dimensional images with the use of said microscope. The sample to be examined is scanned two-dimensionally. In order to generate three-dimensional images, the specimen support and the specimen are moved relative to one another.
The pulse frequency of the non-synchronizable lasers that are of interest for image acquisition especially in the context of multi-photon excitation is preferably in the range from 80 MHz to 90 MHz. The scanning frequency of the scanning system can lie in a range from 40 MHz to 45 MHz.
The pulse frequency of the laser can, however, also be above 100 MHz, and the scanning frequency of the scanning system can be much less than 40 MHz.
Looking at the input circuit of the detection circuit up to the A/D converter, that circuit exhibits a low-pass behavior. When light pulses having a time constant TL are then detected, the signal first rises at the end of the low-pass, and then drops off again (see FIG. 2a). The converter receives a different measurement result depending on the point in time at which the A/D converter converts the voltage value with time constant Ti at its input, despite identical pulse shapes and pulse heights. These errors are superimposed on the actual image brightness signal of the sample, and manifest themselves as a striped pattern in the image (see FIG. 3). Because these artifacts have a certain regularity, they are particularly apparent to the observer as a jitter in the brightness of the structures being examined.
In addition, because of the stroboscope-like illumination of the sample or specimen, only the brightness datum at a specific location in the sample is delivered to the detector. The fluctuation of this observation instant relative to the sampling instant of the A/D converter causes an apparent migration of structures in the region in which the scanned point moves between two A/D converter sampling events. The observer therefore perceives a jitter in the location of the structure being examined.